The proposed research focuses on the biosynthesis of the clinically important antibiotic erythromycin. It is the long-term goal of this project to produce novel erythromycin derivatives that cannot be prepared by traditional chemical syntheses. New antibiotic derivatives such as these are urgently needed, particularly in light of the increased threat posed by newly emergent antibiotic resistant bacteria. In specific, this work seeks to determine the enzymatic domains that dictate the stereochemistry of the erythromycin macrolactone ring and then use this knowledge to genetically engineer the antibiotic producing bacteria, Saccharopolyspora erythraea, to produce new erythromycin derivatives. Currently, the genes for the erythromycin synthase have been cloned, and much is known about the biosynthesis of this chemically complex antibiotic. Yet, the enzymatic domains responsible for the stereochemical configuration of ten distinct sites in the erythromycin macrolactone ring are unknown. Since it has already been shown that the erythromycin synthase can be altered to produce new erythromycin derivatives through genetic engineering, knowledge of the determinants of erythromycin stereochemistry should enable the production of entirely new series of antibiotic derivatives, many of which may be biologically active. Indeed, just through alterations in stereochemistry, over a hundred new erythromycins are theoretically accessible. The approach to be taken here centers initially on the in vitro construction of genetic chimeras encoding altered erythromycin synthases using standard recombinant DNA techniques. These altered synthases will feature enzymatic domain interchanges focusing on those domains most likely involved in the determination of erythromycin stereochemistry (i.e. a domain thought to produce one stereochemical outcome will be replaced with an analogous domain thought to produce the opposite stereochemical outcome). Once the genetic chimeras have been constructed in vitro, the wild type genes of the natural erythromycin-producing organism will be replaced (via a two step gene replacement protocol) and the erythromycin derivatives produced by the mutant organisms will be isolated and characterized by NMR. Importantly, most of the work proposed here will be conducted by undergraduate chemistry and biology majors, consequently this research project will also provide an ideal training opportunity for students interested in medical biotechnology and genetic engineering.